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Notes: These authors used Western analysis to monitor levels of pro- and mature forms of BDNF and NT-3 from the center of lesions after mouse spinal cord injury. Promega Anti-Human NT-3 pAb and Anti-Human BDNF pAb were used to monitor the mature forms of these neurotrophic factors. (3201)

Notes: This paper describes a study of the effect of endogenous neurotrophins on cortical neuron development. Cortical progenitor cells were isolated from mouse embryos and cultured. These precursor cells express both BDNF and NT-3 as well as the corresponding TrkB and TrkC receptors. BDNF and NT-3 signal via Trk receptors to activate the PI3-kinase and MEK pathways. These pathways serve distinct functions, PI3-kinase being essential for progenitor survival and MEK for the differentiation of neurons but not glial cells. The progenitor cell cultures were treated with either Anti-Human BDNF pAb or Anti-Human NT-3 pAb at 20μg/ml to block the function of the endogenous neurotrophins. Anti-ACTIVE® MAPK pAb was used to assess levels of activated MAPK by Western blotting of cell extracts. (2778)

Notes: The effect of oligodendrocytes on rat neuronal cell cultures was studied. The BDNF and NT-3 in oligodendrocyte conditioned medium (CM) were neutralized by blocking with Anti-Human BDNF and Anti-Human NT-3 pAbs. To determine whether BDNF and NT-3 are components of the CM, solutions containing authentic neurotrophins or conditioned medium solutions were preadsorbed at 4°C for 2 hours with neutralizing antibodies and then exposed to the cultures. Control groups received control IgY. Both anti-BDNF and anti-NT-3 were used at 10 μg/ml. This concentration was able to fully block BDNF-induced ChAT activity in cultures, and was able to partially block the effects of the conditioned medium for each antibody. (2826)

Notes: The amounts of neurotrophins NGF, NT-3 and NT-4 were examined in the bladders of Wistar rats at various times after birth up to adulthood. NGF ranged from 5ng per bladder (day 5) to10ng per bladder (day 15). NT-3 ranged from less than 1ng per bladder at postnatal day 30 to as high as about 18ng per bladder in adulthood. NT-4 ranged from about 1ng per bladder on day 30 to more than 40ng per bladder in adulthood. NGF, NT-3, and BDNF protein levels were determined with the NGF Emax® ImmunoAssay System, NT-3 Emax® ImmunoAssay System, and BDNF levels Emax® ImmunoAssay System, respectively. Extraction procedures are detailed. (0228)

Notes: Normal human keratinocytes in culture or conditioned cell media were used for immunoprecipitation of human NT-4 after treatment of the cells with interferon gamma. The immunoprecipitation was performed with the Anti-Human NT-4 pAb and the Immobilized Anti-Chicken IgY. The Anti-Human NT-4 pAb was used for the Western analysis of the immunoprecipitates. Skin biopsies of normal and atopic dermatitis individuals were frozen and cut into 5µm sections. The sections were air dried and acetone fixed. The sections were probed with either the Anti-Human NT-4 pAb (1:400 dilution) or Anti-Human NT-3 pAb (1:400 dilution) followed by colorimetric detection with a HRP-conjugate. (0054)

Notes: NT-4/5,NGF, and neurotrophin-3 (NT-3) was infused into cat primary visual cortex during a period of monocular deprivation. Cats were perfusedtranscardially with PBS, followed by 4% paraformaldehyde. The brain wasembedded in gelatin-albumin in 50-80µm sections. Sections were stained with Promega's Anti-Human NT-4/5, Anti-Human NT-3, or Anti-Human NGF Antibodies to determine the localization of these factors. (2369)

Notes: The Anti-Human NT-3 pAb was used to neutralize NT-3 activity in cultures of Schwann cells. The antibody was also used for immunocytochemistry of cultured Schwann cells and nerves of 7day old rats. The Anti-Human BDNF pAb was also used for its BDNF neutralizing activity but the data was not shown. (0675)

Notes: The NT-3 Emax® ImmunoAssay System was used to quantitate the levels of NT-3 in samples of skin from mice. Excellent detail is provided for tissue processing prior to assay. The Anti-Human NT-3 pAb was also used for immunohistochemistry of 8µm cryostat sections of mouse skin. The factor, Neurotrophin-3, was used for skin organ culture. (1433)

Notes: Conditioned media from rat embryonic inner ear cultures was concentrated and analyzed by Western blotting with the Anti-Human BDNF pAb, Anti-Human NT-3 pAb and the Anti-Rat CNTF pAb. The conditioned media and various amounts of the growth factors were blotted together and developed. The Anti-BDNF pAb, Anti-NT-3 pAb and Anti-CNTF pAb were blotted with 50ng and 10ng of the respective factor along with 50ng of each of the other two factors. The Western analysis detected immunoreactivity only for NT-3 in the conditioned media. Some cross reactivity of the Anti-BDNF pAb for NT-3 and the Anti-NT-3 pAb are reported but clearly each has a preference for the respective factor. Cross reactivity to some level can be expected since both are members of the NGF family of growth factors and the proteins were denatured. As expected, no cross-reactivity with CNTF was observed since it is a different class of growth factor more closely related to cytokines. Inner ear and brain homogenates were analyzed with the BDNF Emax® ImmunoAssay System and the NT-3 Emax® ImmunoAssay System. Inner ear homogenates contained 59pg/ml of BDNF and 320pg/ml NT-3. Brain homogenates produced 440pg/ml BDNF and 436pg/ml NT-3. The BDNF Emax® ImmunoAssay did not recognized NT-3 at levels as high as 600ng/ml and the NT-3 Emax® ImmunoAssay did not recognize BDNF at levels as high as 1ng/ml. (0026)

Notes: The Anti-BDNF pAb and the Anti-NT-3 pAb were used to demonstrate BDNF and NT-3 expression, respectively, in retrovirally transformed human Schwann cells. The immunocytochemistry was observed with anti-chicken IgY-FITC conjugate. The authors also used the Wizard®Plus Minipreps DNA Purification System for plasmid isolation and Promega restriction enzymes for construction of the retroviral vectors. (0446)

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